JP2017532068A - Stable neural stem cells comprising exogenous polynucleotides encoding growth factors and methods of use thereof - Google Patents

Abstract

The present disclosure provides a human neural stem cell comprising an exogenous polynucleotide encoding a growth factor such as IGF-1. Also disclosed are methods of using human neural stem cells for the treatment of neurodegenerative diseases or disorders, including, for example, ALS. The present disclosure relates generally to human neural stem cells comprising exogenous polynucleotides encoding growth factors, including, for example, neurotrophic factors. In one embodiment, the growth factor is stably expressed by human neural stem cells. Such human neural stem cells can be used for the treatment of a neurodegenerative disease or disorder in a subject in need thereof (eg, a human subject having a neurodegenerative disease or disorder).

Description

(Claiming priority)
This application claims priority and benefit under US Provisional Application No. 62 / 147,950 filed on April 15, 2015, and US Provisional Application Number filed October 20, 2014. No. 62 / 066,174 claims priority and benefit, each of which is incorporated herein by reference.

(background)
Because insulin-like growth factor-1 (IGF-1) plays a critical role in cell development and survival in the mammalian central nervous system (CNS), this protein is due to various conditions that affect the CNS. Has been considered a potentially important therapeutic agent. In animal models, delivery of IGF-1 by several methods, including viral vectors and intrathecal injection, has shown promise for the treatment of ALS. However, in clinical trials, subcutaneous administration of mature recombinant IGF-1 to human patients has not demonstrated efficacy in the treatment of ALS. Accordingly, there is a need for improved methods of delivering therapeutically effective amounts of IGF-1 to the site of neuronal loss.

(Summary)
The present disclosure relates generally to human neural stem cells comprising exogenous polynucleotides that encode growth factors including, for example, neurotrophic factors. In one embodiment, the growth factor is stably expressed by human neural stem cells. Such human neural stem cells can be used for the treatment of a neurodegenerative disease or disorder in a subject in need thereof (eg, a human subject having a neurodegenerative disease or disorder).

  The present disclosure provides neural stem cells (eg, stable human neural stem cells) comprising an exogenous polynucleotide encoding insulin-like growth factor 1 (IGF-1). Neural stem cells express IGF-1, including, for example, stable overexpression. A neural stem cell comprising an exogenous polynucleotide encoding IGF-1 surprisingly has a significantly increased number of GAD65 positive GABAergic compared to a neural stem cell not containing an exogenous polynucleotide encoding IGF-1. Resulting in sex neurons.

  The disclosure also provides neural stem cells (eg, stable human neural stem cells) comprising an exogenous polynucleotide encoding a growth factor. Neural stem cells express growth factors, including, for example, stable overexpression.

  In one embodiment of any of the above or below embodiments, the growth factor is insulin-like growth factor 1 (IGF-1), glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF) A neurotrophic factor selected from the group consisting of neurotrophin-3 (NT-3) and vascular endothelial growth factor (VEGF).

  In one embodiment of any of the above or below embodiments, IGF-1 is an IGF-1 isoform, such as IGF-1 isoform 4. In a further embodiment of any of the above or below embodiments, IGF-1 isoform 4 has the nucleotide sequence shown in SEQ ID NO: 1.

  In one embodiment of any of the above or below embodiments, the IGF-1 isoform comprises an N-terminal signal peptide, a mature IGF-1 protein and an E-peptide.

  In one embodiment of any of the above or below embodiments, the human neural stem cell is in a tissue selected from the group consisting of cortex, hippocampus, thalamus, midbrain, cerebellum, hindbrain, spinal cord and dorsal root ganglion. Derived from.

  In one embodiment of any of the above or below embodiments, the human neural stem cell is obtained from a fetus or embryo.

  In one embodiment of any of the above or below embodiments, the human neural stem cells are obtained from a fetus that is about 5 to about 20 weeks old.

  In one embodiment of any of the above or below embodiments, the human neural stem cell is capable of differentiating into neurons and / or glia.

  In one embodiment of any of the above or below embodiments, the human neural stem cell can be engrafted in the brain and / or spinal cord.

  In one embodiment of any of the above or below embodiments, the human neural stem cell is immortalized.

  In one embodiment of any of the above or below embodiments, the human neural stem cell is immortalized by infection with a retrovirus that carries an immortalizing gene.

  In one embodiment of any of the above or below embodiments, the exogenous polynucleotide encoding the growth factor is a ubiquitin C (UbC) promoter (eg, ubiquitin C (UbC) having the nucleotide sequence set forth in SEQ ID NO: 3. ) Promoter), human phosphoglycerate kinase 1 promoter, human synapsin promoter or synthetic CAG promoter.

  The disclosure also includes a human neural stem cell comprising an exogenous polynucleotide encoding insulin-like growth factor 1 (IGF-1), wherein IGF-1 comprises the nucleotide sequence set forth in SEQ ID NO: 1, and an IGF-1 nucleotide Human neural stem cells in which the sequence is stably expressed are provided.

  In one embodiment of any of the above or below embodiments, the human neural stem cell is immortalized.

  In one embodiment of any of the above or below embodiments, the exogenous polynucleotide encoding IGF-1 is a ubiquitin C (UbC) promoter (eg, ubiquitin C having the nucleotide sequence set forth in SEQ ID NO: 3 ( UbC) promoter), human phosphoglycerate kinase 1 promoter, human synapsin promoter or synthetic CAG promoter.

  The present disclosure is also a method for the treatment of a neurodegenerative disease or disorder, wherein the subject (eg, a human subject having a neurodegenerative disease or disorder) has an exogenous encoding an IGF-1 in a therapeutically effective amount. There is provided a method comprising administering one or more neural stem cells (eg, stable human neural stem cells) comprising a sex polynucleotide.

  The disclosure is also a method for the treatment of a neurodegenerative disease or disorder, wherein the subject (eg, a human subject having a neurodegenerative disease or disorder) exogenously encodes a therapeutically effective amount of a growth factor. There is provided a method comprising administering one or more neural stem cells (eg, stable human neural stem cells) comprising a polynucleotide.

  In one embodiment of any of the above or below embodiments, the growth factor is insulin-like growth factor 1 (IGF-1), glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF) A neurotrophic factor selected from the group consisting of neurotrophin-3 (NT-3) and vascular endothelial growth factor (VEGF).

  In one embodiment of any of the above or below embodiments, IGF-1 is an IGF-1 isoform, such as IGF-1 isoform 4. In a further embodiment of any of the above or below embodiments, IGF-1 isoform 4 has the nucleotide sequence shown in SEQ ID NO: 1.

  In one embodiment of any of the above or below embodiments, the IGF-1 isoform comprises an N-terminal signal peptide, a mature IGF-1 protein and an E-peptide.

  In one embodiment of any of the above or below embodiments, the therapeutically effective amount of one or more human neural stem cells is capable of differentiating into neurons and / or glia.

  In one embodiment of any of the above or below embodiments, a therapeutically effective amount of one or more human neural stem cells can be engrafted in the brain or spinal cord.

  In one embodiment of any of the above or below embodiments, the exogenous polynucleotide encoding the growth factor is a ubiquitin C (UbC) promoter (eg, ubiquitin C (UbC) having the nucleotide sequence set forth in SEQ ID NO: 3. ) Promoter), human phosphoglycerate kinase 1 promoter, human synapsin promoter or synthetic CAG promoter.

  In one embodiment of any of the above or below embodiments, the neurodegenerative disease or disorder is amyotrophic lateral sclerosis (ALS), spinal cord injury (SCI), traumatic brain injury (TBI), Alzheimer's. Disease (AD), dementia, mild cognitive impairment, diabetes, diabetes-related CNS complications, peripheral neuropathy, retinal neuropathy or multiple sclerosis.

  In one embodiment of any of the above or below embodiments, the spinal cord injury is traumatic spinal cord injury or ischemic spinal cord injury.

  In one embodiment of any of the above or below embodiments, a therapeutically effective amount of one or more neural stem cells is injected into the area of neurodegeneration.

  In one embodiment of any of the above or below embodiments, a therapeutically effective amount of one or more neural stem cells is administered at about 5 to about 50 sites in the area of neurodegeneration.

  In one embodiment of any of the above or below embodiments, the one or more sites are separated by a distance of approximately 100 microns to about 5000 microns.

  In one embodiment of any of the above or below embodiments, at least 20%, 30%, 40%, 50%, 60%, 70%, 80% of a therapeutically effective amount of one or more neural stem cells. 90%, 95% or more can generate neurons in the area of neurodegeneration.

  In one embodiment of any of the above or below embodiments, the subject is a human.

  The disclosure also includes a method of making a human neural stem cell comprising an exogenous polynucleotide encoding a growth factor, wherein the growth factor is stably expressed, obtaining one or more human neural stem cells; Plating one or more neural stem cells on a tissue culture treated dish pre-coated with D-lysine and fibronectin and culturing the one or more neural stem cells in a growth medium (eg, serum-free growth medium) A method comprising: expanding one or more neural stem cells to produce a population of expanded neural stem cells; and infecting the neural stem cells with a vector encoding a growth factor. In one embodiment, expanded neural stem cells are immortalized by infecting the expanded neural stem cells with a retrovirus encoding an immortalizing gene.

  In one embodiment of any of the above or below embodiments, the growth factor is insulin-like growth factor 1 (IGF-1), glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF) A neurotrophic factor selected from the group consisting of neurotrophin-3 (NT-3) and vascular endothelial growth factor (VEGF).

  In one embodiment of any of the above or below embodiments, IGF-1 is an IGF-1 isoform, such as IGF-1 isoform 4. In a further embodiment of any of the above or below embodiments, IGF-1 isoform 4 has the nucleotide sequence shown in SEQ ID NO: 1.

  In one embodiment of any of the above or below embodiments, the IGF-1 isoform comprises an N-terminal signal peptide, a mature IGF-1 protein and an E-peptide.

  In one embodiment of any of the above or below embodiments, human neural stem cells are obtained from tissue isolated post mortem from an aborted human fetus.

  In one embodiment of any of the above or below embodiments, human neural stem cells are infected with a replication defective retrovirus having a copy of the Myc-ER fusion gene.

  The present disclosure is a method of reducing amyloid beta (Aβ) deposition in a subject's brain, eliminating Aβ deposits in a subject's brain (eg, hippocampus and / or cortex), or preventing Aβ accumulation in a subject's brain. Providing a therapeutically effective amount of one or more human neural stem cells comprising an exogenous polynucleotide encoding IGF-1 to one or more regions of the subject's brain.

  The present disclosure is a method of increasing the number of cholinergic neurons in a subject's brain (eg, hippocampus and / or cortex), wherein a therapeutically effective amount of IGF-1 is applied to one or more regions of the subject's brain. A method comprising administering one or more human neural stem cells comprising an exogenous polynucleotide encoding.

  The present disclosure is also a method of repairing synapses in a subject's brain, comprising one or more exogenous polynucleotides encoding IGF-1 in one or more regions of the subject's brain. A method is provided comprising administering human neural stem cells.

  The present disclosure is a method for restoring memory and / or cognition in a subject comprising a therapeutically effective amount of an exogenous polynucleotide encoding IGF-1 in one or more regions of the subject's brain 1 Or a method comprising administering a plurality of human neural stem cells.

  The foregoing summary, as well as the following detailed description of the disclosure, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the present disclosure, presently preferred embodiments are shown in the drawings. However, it should be understood that this disclosure is not limited to the precise arrangements, examples, and instrumentalities shown.

IGF-I production and signaling in HK532 and HK532-IGF-I cells. (A) IGF-I production in HK532 and HK532-IGF-I through early differentiation. (B) Representative ICC images of D7 HK532 and HK532-IGF-I labeled with DAPI (blue) and IGF-IR (green). Scale bar 50 μm. (C) Undifferentiated and differentiated (D7) Western blot analysis of IGF-I signaling in HK532 and HK532-IGF-I. Cells were treated for 1 hour with a panel of LY, U or NVP inhibitors and IGF-I treatment was continued for 30 minutes. All blots were probed with pIGF-IR, IGF-IR, pERK, ERK, pAKT and AKT. Β-actin was used as a loading control.

Induced IGF-I expression does not affect HK532 proliferation and migration. (A) Quantification of the percentage of EdU positive cells at D0, D3 and D7 in HK532 and HK532-IGF-I cultures. (B-E) Representative ICC images of D0 and D7 HK532 and HK532-IGF-I labeled with DAPI (blue) and EdU (green). Scale bar 200 μm. (FG) Quantification of absorbance of migrated HK532 and HK532-IGF-I at D0 and D7.

Induced IGF-I expression does not affect maintenance of progenitor cell state during differentiation or neurite outgrowth. (AB) Representative ICC images of D0 HK532 and HK532-IGF-I labeled with DAPI (blue) and Nestin (red). (C) Quantification of Nestin positive D0 HK532 and HK532-IGF-I. (DE) Representative ICC images of D7 HK532 and HK532-IGF-I labeled with DAPI (blue) and TUJ1 (red). (F) Quantification of nerve index measurements (μm ² / cell). Scale bar 200 μm.

Final phenotype of HK532 and HK532-IGF-I cells. (AB) Representative ICC images of D7 HK532 and HK532-IGF-I cells labeled with DAPI (blue) and GAD65 (green). (C-D) Representative ICC images of D7 cells labeled with DAPI (blue) and VGLUT (red). Scale bar 200 μm. (E) Quantification of GAD65 positive GABAergic neurons in HK532 and HK532-IGF-I cells. HK532-IGF-I cells preferentially differentiate into GABAergic neurons (* p <0.05). (F) Quantification of VGLUT positive glutamatergic neurons in HK532 and HK532-IGF-I cells.

FIG. HK532-IGF-I cells are neuroprotective and survival grafted to APP / PS1 AD and WT mice. (A) Quantification of apoptosis and CC3 activation in response to Aβ toxicity in primary CN, HK532 and HK532-IGF-I. Both HK532 cell lines are more resistant than CN (* p <0.05). (BD) Representative ICC images of primary CN labeled with DAPI and CC3 (scale bar 200 μm). (B) Control CN without Aβ treatment, (C) CN with Aβ treatment, (D) CN with Aβ treatment co-cultured with HK532 and (E) Aβ treatment co-cultured with HK532-IGF-I CN which gave. (F) Quantification of Aβ-mediated apoptosis and CC3 activation in CN / HK532 co-culture. HK532-IGF-I showed increased neuroprotective capacity compared to HK532 (* p <0.05). (GH) Representative DAPI, HuNu and DCX labeling of human early neural progenitors in the hippocampal region of (G) APP / PS1 AD and (H) WT animals 10 weeks after transplantation into hippocampal forebrain arch Images (10 × scale bar 200 μm; 60 × scale bar 50 μm). FIG. HK532-IGF-I cells are neuroprotective and survival grafted to APP / PS1 AD and WT mice. (A) Quantification of apoptosis and CC3 activation in response to Aβ toxicity in primary CN, HK532 and HK532-IGF-I. Both HK532 cell lines are more resistant than CN (* p <0.05). (BD) Representative ICC images of primary CN labeled with DAPI and CC3 (scale bar 200 μm). (B) Control CN without Aβ treatment, (C) CN with Aβ treatment, (D) CN with Aβ treatment co-cultured with HK532 and (E) Aβ treatment co-cultured with HK532-IGF-I CN which gave. (F) Quantification of Aβ-mediated apoptosis and CC3 activation in CN / HK532 co-culture. HK532-IGF-I showed increased neuroprotective capacity compared to HK532 (* p <0.05). (GH) Representative DAPI, HuNu and DCX labeling of human early neural progenitors in the hippocampal region of (G) APP / PS1 AD and (H) WT animals 10 weeks after transplantation into hippocampal forebrain arch Images (10 × scale bar 200 μm; 60 × scale bar 50 μm).

Aβ levels are significantly reduced when HK532-IGF-I transplantation is applied. Fluorescence microscopy shows separate formation of (A-C) and (DF) Aβ plaques (arrows) in the hippocampus of APP / PSI vehicle-treated mice. Plaques were visibly reduced when HK532-IGF-I treatment was applied. In all sections nuclei were stained with DAPI (blue). Scale bar: 100 μm. (G) Quantification of fluorescence intensity (AU) change from non-tg of vehicle-treated and NSC-treated groups showed significantly reduced levels of Aβ in HK532-IGF-I treated mice versus sham Suggests that HK532-IGF-I mediates Aβ accumulation. *** P <0.001. (H) Specifically, comparison of Aβ fluorescence levels in the cortex and hippocampus shows a significant reduction in Aβ deposition in cortical sections of mice injected with NSC compared to vehicle injected mice, but ** * P <0.001, with hippocampal slices, the decreased level was not significant.

HK532-IGF-I engraftment rescues cholinergic neurons in the striatum. (AC) Fluorescent images of striatum immunostained for ChAT (green) in each group. Scale bar 100 μm. (D) High magnification image of ChAT positive cells. Nuclei were stained with DAPI (blue). Scale bar: 50 μm. (E) Cells in the striatum of all mice showed significant loss of cholinergic neurons in vehicle / injected APP / PSI mice compared to non-tg mice. * P <0.05. (F) Calculation of fold change of cholinergic neurons from non-tg mice shows significantly higher amounts in APP / PSI mice treated with HK532-IGF-I. * P <0.05.

HK532-IGF-I increases presynaptic activity and forms synapses with endogenous neurons. (AC) Fluorescence microscopy of hippocampal slices stained for synaptophysin visually shows fluorescence signal in non-tg and NSC injected APP / PSI mice compared to vehicle injected APP / PSI mice Increased strength is indicated. Scale bar: 100 μm. (DF) High-magnification images of synaptophysin near the granule cell layer of the dentate gyrus. Scale bar: 50 μm. (GJ) Fluorescent images of sections from NSC-treated groups immunostained for human NuMA (red), synaptophysin (green) and DAPI (blue) showing presynaptic function around NSC. Scale bar: 50 μm.

Frozen immunohistochemical analysis showing the presence of human cell grafts in the anterior horn and a broad distribution in gray and white matter (FIGS. 9a-c). In each image, SC121 (green) indicates all human cytoplasm and DAPI (blue) indicates all cell nuclei. The inset in A is indicated by A ', and the inset in A' is indicated by A ".

(Detailed explanation)
The present disclosure relates to a neural stem cell (eg, a human neural stem cell derived from a fetus or embryo) that includes an exogenous polynucleotide encoding a growth factor including, for example, a neurotrophic factor, wherein the growth factor is stably expressed by the neural stem cell A neural stem cell is provided. The inventors have surprisingly found that neural stem cells engraft at the site of neuronal loss and stably express growth factors, including neurotrophic factors such as mature IGF-1, in a therapeutically effective amount. I found it possible. Examples of neurotrophic factors include insulin-like growth factor 1 (IGF-1) (for example, IGF-1 isoform having the sequence shown in SEQ ID NO: 1), glial cell line-derived neurotrophic factor (GDNF), brain-derived nerve Mention may be made of trophic factor (BDNF), neurotrophin-3 (NT-3) or vascular endothelial growth factor (VEGF). However, any protein that can be secreted by neural stem cells for use in the present disclosure is contemplated. Such human neural stem cells can be obtained from neural stem cell lines, including but not limited to amyotrophic lateral sclerosis (ALS), spinal cord injury (SCI), traumatic brain injury (TBI), Alzheimer's disease (AD), treatment of neurodegenerative diseases or disorders including various CNS indications including dementia, mild cognitive impairment, diabetes, diabetes-related CNS complications, peripheral neuropathy, retinal neuropathy and multiple sclerosis Can be used for.

  Surprisingly, the inventors include an exogenous polynucleotide that encodes IGF-1, wherein IGF-1 is stably expressed by human neural stem cells, wherein the human neural stem cell is an exogenous that encodes IGF-1. It has been discovered that neural stem cells that do not contain sex polynucleotides result in a significantly increased number of GAD65-positive GABAergic neurons (ie, that can differentiate into it and / or support its growth). Since degeneration specific to GABAergic neurons has been reported in mouse models and human patients, this is therapeutically associated with Alzheimer's disease (Loreth et al. (2012) Neurobiol Dis, 2012, 47 ( 1): 1-12; Schwab et al. (2013), J Alzheimers Dis, 2013, 33 (4): 1073-88). Thus, transplantation of neural stem cells that stably express IGF-1 provides a source of de novo GABAergic neurons, replacing those selectively lost in Alzheimer's disease, which are important neural networks in the brain To repair.

  The neural stem cells of the present disclosure that express growth factors can be stable and multipotent because they efficiently engraft in the brain and spinal cord, differentiate simultaneously into neurons and glia, and are incorporated into host tissues. Such integration involves the formation of synaptic connections between host neurons and grafted stem cell-derived neurons. The advantage of stable engraftment and integration in the CNS is that cell grafting and thereby production of growth factors can be constant and stable as long as the cells are alive. Furthermore, the formation of synaptic connections between host neurons and grafted neurons allows direct delivery of growth factors to the synapses and interstitial spaces adjacent to damaged or diseased neurons. In addition, neural stem cells themselves are replaced by neurons that are therapeutic and produce various known growth factors that can be lost towards the disease process.

  Furthermore, the neural stem cells of the present disclosure provide the advantage that their glial progeny migrate widely in the brain and spinal cord, while their neuronal progeny remain localized near the site of injection. This property makes it possible to selectively target either localized delivery of growth factors by neurons or widely distributed delivery in the CNS by glia. The advantage of neuronal secretion of growth factors such as neurotrophic factors such as IGF-1, GDNF, BDNF, NT3, NGF, VEGF, etc. is the limited space of the synaptic cleft immediately adjacent to high concentrations of the corresponding receptors. Including, even small amounts of growth factors can reach therapeutic doses because they can be continuously released into the extracellular space adjacent to the target cell, and immediately taken up by the target cell and transported retrogradely (Rind et al. (2005), J Neurosci, 25: 539-549).

  Further disclosed are neural stem cells capable of producing a desired proportion of neurons and / or glia (eg, producing 60% neurons and 40% glia) as a result of their origin or their growth conditions. These neural stem cells that produce a high percentage of neurons can be used to deliver growth factors locally to specific target areas as needed, whereas those that produce a high percentage of glia It can be used to deliver growth factors more globally. Examples of growth factors that may be desirable to administer locally include, but are not limited to, neurotrophic factors such as IGF-1, NGF, NT3 or BDNF that have a multidirectional effect. Examples of growth factors that may be desirable to administer comprehensively include, but are not limited to, proteins for enzyme replacement, such as for the treatment of lysosomal disease, monoclonal antibodies to cytokines, cytokine receptors or growth factors A receptor.

  In one embodiment, the growth factor is a neurotrophic factor such as IGF-1, including, for example, the IFG1 isoform shown in FIG. The IGF-1 isoform can be IGF-1 isoform 4. In one embodiment, IGF-1 isoform 4 has the nucleotide sequence shown in SEQ ID NO: 1 (amino acid sequence shown in SEQ ID NO: 2). This isoform is composed of three different potential biological effectors: various IGF binding proteins as well as mature IGF-1 protein capable of binding to IGF-1 receptor; by a mechanism independent of IGF-1 receptor, Carboxy (C-) terminal MGF peptide released during pro-IGF-1 processing that can act as a neuroprotective agent against ischemia and other adverse conditions; and released during pre-pro-IGF-1 processing Containing an amino (N-) terminal signal peptide.

  IGF-1 biology is complex. Six different forms of human IGF-1 mRNA transcripts were produced under the control of two different promoters (reviewed in Barton (2006), J Appl Physiol, 100: 1778-1784), All of which produce a single mature IGF-1 protein. The various transcripts are translated into mature IGF-1 protein, during which time separate cleavage products are produced. Transcript isoforms and various cleavage products are known to be tissue specific. Thus, 75% of circulating mature IGF-1 protein is produced by the liver, but several other tissues, including muscle, kidney and brain / spinal cord, produce their own IGF-1 transcripts and proteins. To do. Also, IGF-1 levels in the brain are regulated independently of, for example, plasma levels of IGF-1 (Adams et al. (2009), Growth Factors, 27: 181-188). In particular, rat and mouse IGF-1 genes are regulated differently than human IGF-1 genes, resulting in unequal IGF-1 isoforms between species (Barton (2006), Appl. Physiol. Nutr. Metab). 31, 791-797).

  The growth factor dose (eg, therapeutically effective dose) and localization in the CNS can be altered by using different promoters and by using different neural stem cell lines with distinct differentiation and migration characteristics. . For example, the synapsin promoter drives expression primarily at low to medium levels in neuronal progeny, and thus can be used to ensure a localized distribution targeting the neuronal population. In contrast, the ubiquitin C promoter can be used to drive expression to both neurons and glial progeny, allowing for a wider distribution of growth factors. Furthermore, a synthetic CAG promoter consisting of a cytomegalovirus (CMV) enhancer fused to a chicken β-actin promoter can direct very high levels of expression of growth factors. In addition, if necessary, neural stem cells that produce a higher percentage of neurons can be used to deliver growth factors locally to a specific target area, whereas neural stem cells that produce a higher percentage of glia It can be used to deliver growth factors more comprehensively. Examples of growth factors that may be desired to be administered locally include, but are not limited to, neurotrophic factors such as IGF-1, NGF, NT3 or BDNF that have a multidirectional effect. Examples of growth factors that may be desired to be administered comprehensively include, but are not limited to, proteins for enzyme replacement such as for the treatment of lysosomal disease, monoclonal antibodies to cytokines, cytokine receptors or growth factor receptors Is mentioned.

(Neural stem cells)
Neural stem cells (eg, stable human neural stem cells) comprising an exogenous polynucleotide encoding a growth factor such as a neurotrophic factor are provided. Neurotrophic factors include insulin-like growth factor 1 (IGF-1), glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) or vascular endothelial cells It can be a growth factor (VEGF). Also provided are neural stem cell lines comprising neural stem cells having exogenous polynucleotides that encode growth factors. Neural stem cells are preferably stable and do not differentiate in culture even after more than 60 cell doublings.

  The present disclosure provides neural stem cells (eg, stable human neural stem cells) comprising an exogenous polynucleotide encoding insulin-like growth factor 1 (IGF-1). Neural stem cells express IGF-1, including, for example, stable overexpression. A neural stem cell comprising an exogenous polynucleotide encoding IGF-1 surprisingly has a significantly increased number of GAD65 positive GABA compared to a neural stem cell not containing an exogenous polynucleotide encoding IGF-1. Results in operative neurons. Neural stem cells are preferably stable and do not differentiate in culture even after more than 60 cell doublings.

  The present disclosure provides stable human neural stem cells that express an exogenous polynucleotide encoding insulin-like growth factor 1 (IGF-1).

  In one embodiment, the neurotrophic factor is IGF-1, including an IGF-1 isoform such as, for example, IGF-1 isoform 4 having the sequence shown in SEQ ID NO: 1. Surprisingly, the inventors have shown that IGF-1 isoform 4 having the sequence shown in SEQ ID NO: 1 is functional when expressed by neural stem cells (eg, binds to its receptor, Initiate a signaling process with a physiological impact). Several previous reports indicate that administration of neural stem cells expressing mature IGF-1 is not effective in providing a functional benefit (ie, neural stem cells are not effective in treating a disease or disorder). As such, this finding was completely unexpected.

  As used herein, the term “neural stem cell” or “NSC” refers to differentiation into each of the three major cell types of the central nervous system (CNS): neurons, astrocytes and oligodendrocytes. Refers to pluripotent stem cells that can be functionally defined according to their ability to As used herein, the term “stem cell” refers to an undifferentiated cell that is capable of self-renewal, meaning that for each cell division, at least one daughter cell is also a stem cell. NSCs can also refer to neural or neuronal progenitor cells or neuroepithelial progenitors.

  The disclosure also includes a method of making a human neural stem cell comprising an exogenous polynucleotide encoding a growth factor, wherein the growth factor is stably expressed, obtaining one or more human neural stem cells; Plating one or more neural stem cells on a tissue culture treated dish pre-coated with D-lysine and fibronectin, culturing one or more neural stem cells in a serum-free growth medium, 1 or Proliferating multiple neural stem cells to produce a population of expanded neural stem cells, infecting the proliferated neural stem cells with a retrovirus encoding an immortalizing gene, and a neural stem cell previously infected with a retrovirus Infecting with a vector encoding a growth factor. Such immortalized neural stem cells and methods for producing neural stem cells are disclosed in US Pat. No. 7,544,511.

  In one embodiment, the NSC is pluripotent so that each cell has the ability to differentiate into a neuron, astrocyte or oligodendrocyte. In another embodiment, the NSCs are bipotent so that each cell has the ability to differentiate into two of the three cell types of the CNS. In another embodiment, the NSC comprises at least a bipotent cell that generates both neurons and astrocytes in vitro, and at least a unipotent cell that generates neurons in vivo.

  Growth conditions can affect the direction of differentiation of a cell towards one cell type or another, indicating that the cell is not tilted towards a single lineage. In culture conditions that favor neuronal differentiation, especially cells derived from the human CNS are mostly bipotential towards neurons and astrocytes, and differentiation into oligodendrocytes is minimal. Thus, differentiated cell cultures of the disclosed method can give rise to neurons and astrocytes.

  In one embodiment, the NSC is isolated from the CNS. As used herein, the term “isolated” with respect to a cell means that the cell is naturally occurring (eg, the cell is naturally present in an organism) and the cell is removed from its natural environment. Refers to a cell in a different environment.

  NSCs can be isolated from regions that are naturally neurogenic for the desired neuronal population and from embryos, fetuses, postnatal, juvenile or adult tissues. Is the desired population of cells lost in the course of disease progression? Or it may include cells of a particular neuronal phenotype that can be replaced or supplemented by such an inactive phenotype. In one embodiment, NSCs are isolated from the subventricular zone (SVZ) or from the subcellular zone of the dentate gyrus (DG). In a preferred embodiment, NSCs are isolated from spinal cords in which ventral motor neuron neurogenesis is present and obtained in embryonic human fetal development in which ventral motor neuron neurogenesis is present.

  Thus, in one embodiment, NSCs are isolated from the spinal cord at about 6.5 to about 20 weeks of gestational age. Preferably, NSCs are isolated from the spinal cord at about 7 to about 9 weeks of gestational age. In another embodiment, the NSC is isolated from embryonic spinal cord tissue. In yet another embodiment, the neural stem cell is isolated from a human. It should be understood that the percentage of NSC population that can be isolated can vary with the age of the donor. The proliferative capacity of the cell population can also vary with the age of the donor.

  The ventral midbrain NSC is different from, for example, NSCs obtained from the spinal cord of the same pregnancy stage. In particular, NSCs derived from the ventral midbrain can generate dopaminergic neurons that express tyrosine-hydroxylase, whereas NSCs derived from the spinal cord can generate acetylcholine-producing cholinergic neurons. However, both cell types simultaneously produce more ubiquitous glutamate and GABA producing neurons. Thus, in one embodiment, the disclosed method obtains NSCs from the spinal cord to treat a condition that is ameliorated or attenuated by at least some implantation of acetylcholine producing cholinergic neurons. Including that.

  NSCs can also be isolated from postnatal and adult tissues. NSCs derived from postnatal and adult tissues are quantitatively equivalent in terms of their ability to differentiate into neurons and glia and in their growth and differentiation characteristics. However, the efficiency of in vitro isolation of NSCs from various postnatal and adult CNS can be significantly lower than that of NSCs from fetal tissue with a richer population of NSCs. Nonetheless, the disclosed method allows at least about 30% of NSCs derived from neonatal and adult sources to differentiate into neurons in vitro, as well as fetal-derived NSCs. Thus, postnatal and adult tissue can be used as described above in the case of fetal-derived NSCs.

  In one embodiment, human fetal spinal cord tissue is dissected under a microscope. A region of tissue corresponding to the lower cervical / upper breast segment is isolated. NSCs are isolated, pooled, and grown on poly-D-lysine coated culture vessels in medium containing fibronectin and basic fibroblast growth factor (bFGF; FGF-2). The cells are grown and then concentrated to a desired target cell density of about 10,000 cells per microliter in medium without preservatives and antibiotics. The concentrated cells may be used fresh for implantation or frozen for later use.

  In one embodiment, the NSCs are derived from embryonic stem cells or induced pluripotent stem cells. As used herein, the term “embryonic stem cell” refers to a developing embryo that can give rise to all of the cells of the body (eg, cells of the outer-, middle- and / or endoderm cell lineage). Refers to isolated stem cells. The term “induced pluripotent stem cell” as used herein refers to a somatic cell-derived stem cell (eg, a differentiated somatic cell) that has a higher capacity than a somatic cell. Embryonic stem cells and induced pluripotent stem cells can differentiate into more mature cells (eg, neural stem cells or neural progenitor cells). Methods used to grow and differentiate embryonic or induced pluripotent stem cells into NSCs in vitro are described, for example, by Daadi et al., PLoS One., 3 (2): e1644 (2008). And the like.

  There are a number of standard molecular biology techniques that can be used to modulate the expression of a polynucleotide encoding a growth factor in neural stem cells as disclosed herein. For example, different promoters can be used to regulate the level of expression of growth factors and / or which progeny of neural stem cells express the factor. For example, the human ubiquitin C (UbC), PGK, or CAG promoter confers distinct levels of expression of growth factors in the differentiated neurons and glial progeny of the human neural stem cells disclosed herein. The PGK promoter allows low levels of growth factor production, approximately 0.5 ng of protein per million cells per 24 hours, and the UbC promoter produces large amounts of growth factor, per million cells per 24 hours. Allowing approximately 2 ng of protein, the CAG promoter also allows for higher amounts of growth factor production, approximately 14 ng of protein per million cells per 24 hours. Additionally or alternatively, expression can be driven and restricted to specific progeny of neural stem cells. For example, the human synapsin promoter can be used to direct growth factor expression to neuronal progeny of neural stem cells.

(Method of treatment)
Neural stem cells as disclosed herein are amyotrophic lateral sclerosis (ALS), spinal cord injury (SCI), traumatic brain injury (TBI), Alzheimer's disease (AD), dementia, mild cognitive impairment Can be used in a method for treating a disease or disorder, including a neurodegenerative disease or disorder, such as diabetes, diabetes-related CNS complications, peripheral neuropathy, retinal neuropathy or multiple sclerosis. Such methods can include administering to a subject a therapeutically effective amount of neural stem cells disclosed herein, including, for example, by injection. In one embodiment, a subject treated with the disclosed neural stem cells is immunosuppressed before, during and / or after administration of neural stem cells.

  In some embodiments, “treating” or “treatment” of a disease, disorder or condition, at least in part, (1) preventing the disease, disorder or condition, ie, to the disease, disorder or condition. Preventing clinical manifestations of a disease, disorder or condition from occurring in a mammal that has been exposed or has a predisposition but has not yet experienced or shown symptoms of the disease, disorder or condition; (2 ) Inhibiting a disease, disorder or condition, ie, stopping or reducing the occurrence of a disease, disorder or condition or clinical symptoms thereof, or (3) reducing a disease, disorder or condition, ie disease, disorder Or withdrawing the condition or its clinical symptoms. A neurodegenerative disease or disorder is treated when a subject to whom the disclosed neural stem cells are administered exhibits an improvement in hippocampal-dependent behavioral problems compared to a subject that is not treated with the disclosed neural stem cells. Can be considered.

  The terms “prevent”, “prevent”, “prevent”, “suppress”, “suppress”, “suppress”, “inhibit” and “inhibit” as used herein, Prevent, suppress or reduce the occurrence of clinical signs of a disease state or condition (eg, the formation of Aβ deposits), either temporarily or permanently (eg, prior to the occurrence of clinical symptoms of the disease state or condition such as Aβ deposits) Refers to the course of action initiated by such a method (such as administering an NSC as disclosed herein). Such prevention, suppression or reduction is not necessarily absolute in order to be useful.

  In some embodiments, an “effective amount” as used herein refers to the amount of spinal cord-derived neural stem cells that is necessary to confer a therapeutic effect on a subject. A “therapeutically effective amount” as used herein refers to a sufficient amount of spinal cord-derived neural stem cells being administered that reduces to some extent one or more symptoms of the disease, disorder or condition being treated. Point to. In some embodiments, the result is a reduction and / or alleviation of the cause of a sign, symptom or disease or any other desired change in the biological system. For example, in some embodiments, an “effective amount” for therapeutic use is an amount of spinal cord-derived neural stem cells required to provide a clinically significant reduction in disease symptoms without undue adverse side effects. Amount. In some embodiments, for any individual, an appropriate “effective amount” is determined using techniques such as a dose escalation study. The term “therapeutically effective amount” includes, for example, a prophylactically effective amount. In other embodiments, an “effective amount” of spinal cord-derived neural stem cells is an amount effective to achieve the desired pharmacological effect or therapeutic improvement without undue adverse side effects. In other embodiments, an “effective amount” or “therapeutically effective amount” is a variation in metabolism, age, weight, subject general condition, condition being treated, severity of condition being treated and judgment of the prescribing physician. It is understood that for each subject changes.

  Neural stem cells can be transplanted in motor cortex and / or spinal cord gray to rescue degenerated upper and lower motor neurons in ALS, or affected in the acute and chronic stages of ischemic or hemorrhagic stroke Can be transplanted into the site of infarction to rescue neurons and reduce border size, and in the Meinert basal ganglia to protect cholinergic neurons in patients with dementia and Alzheimer's disease Can be transplanted into the hippocampus or other areas of the brain to slow down the progression of dementia, or to reduce seizures in epilepsy during aging or in Alzheimer's disease, White as encapsulated and corpus callosum for neuroprotection in traumatic brain injury or in stroke It can be implanted in the region. Neural stem cells can migrate rapidly from these locations into the brain and distribute IGF-1 protein for the treatment of other indications such as diabetes and diabetes-related CNS complications. Neural stem cells can be transplanted into the intercostal and / or diaphragm muscles to increase muscle endplate and enhance the respiratory capacity of patients with ALS or cervical spinal cord injury. Neural stem cells can be transplanted into skeletal muscle to increase muscle fiber in muscular dystrophy and various motor neuron diseases. Neural stem cells can be transplanted into the cerebellum and / or brainstem to rescue motor neurons affected by conditions including spinal muscular atrophy, medullary muscular atrophy and cerebellar ataxia. Neural stem cells can be transplanted into the spinal cord for regeneration of myelinated oligodendrocytes in multiple sclerosis. Neural stem cells can be transplanted into the intrathecal space or into the subarachnoid space for global distribution of IGF-1 for neuroprotection in enzyme deficiency diseases.

  The present disclosure relates to a method of reducing amyloid beta (Aβ) deposition (eg, the level of Aβ deposition) in a subject's brain (eg, hippocampus and / or cortex), comprising: There is provided a method comprising administering a therapeutically effective amount of one or more human neural stem cells comprising an exogenous polynucleotide encoding IGF-1. The level of Aβ in the subject's brain is 5%, including a comparison with a subject's brain that has not been administered a therapeutically effective amount of one or more human neural stem cells comprising an exogenous polynucleotide encoding IGF-1. It can be reduced by 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200% or more. The level of Aβ in the subject's brain is doubled, including a comparison with a subject's brain that has not been administered a therapeutically effective amount of one or more human neural stem cells comprising an exogenous polynucleotide encoding IGF-1. It can be reduced by 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more. In some embodiments, the subject has Alzheimer's disease.

  The present disclosure is a method for eliminating Aβ deposits in a subject's brain (eg, hippocampus and / or cortex) or preventing Aβ accumulation in a subject's brain (eg, hippocampus and / or cortex), comprising: Administering one or more human neural stem cells comprising an exogenous polynucleotide encoding IGF-1 to one or more regions of the brain. In some embodiments, the subject has Alzheimer's disease.

  The present disclosure is a method for preventing Aβ accumulation in a subject's brain (eg, hippocampus and / or cortex), wherein the exogenous encoding a therapeutically effective amount of IGF-1 in one or more regions of the subject's brain. A method is provided comprising administering one or more human neural stem cells comprising a sex polynucleotide. In some embodiments, the subject has Alzheimer's disease.

  The present disclosure is a method of increasing the number of cholinergic neurons in a subject's brain (eg, hippocampus and / or cortex), wherein a therapeutically effective amount of IGF-1 is applied to one or more regions of the subject's brain. A method comprising administering one or more human neural stem cells comprising an exogenous polynucleotide encoding. The number of cholinergic neurons in the subject's brain includes a comparison with a subject's brain that has not been administered a therapeutically effective amount of one or more human neural stem cells comprising an exogenous polynucleotide encoding IGF-1. May be increased by 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200% or more . The number of cholinergic neurons in the subject's brain includes a comparison with a subject's brain that has not been administered a therapeutically effective amount of one or more human neural stem cells comprising an exogenous polynucleotide encoding IGF-1. It can be increased by 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. In some embodiments, the subject has Alzheimer's disease.

  The present disclosure is also a method of repairing synapses in a subject's brain, comprising one or more exogenous polynucleotides encoding IGF-1 in one or more regions of the subject's brain. A method is provided comprising administering human neural stem cells. In some embodiments, the subject has Alzheimer's disease.

  The present disclosure is a method for repairing a subject's memory and / or cognition, the subject's brain of one or more human neural stem cells comprising a therapeutically effective amount of an exogenous polynucleotide encoding IGF-1. A method comprising administering to one or more regions. In some embodiments, the subject has Alzheimer's disease.

  In one embodiment, the NSC can be diluted with an acceptable drug carrier. The term “pharmaceutically acceptable carrier” as used herein with which the cells of the disclosure are administered and approved by a federal or state government regulatory authority, or in an animal, More specifically, it refers to diluents, adjuvants, excipients or vehicles listed in the US Pharmacopoeia or other commonly recognized pharmacopoeia for use in humans. Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical carrier can be saline, gum arabic, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. When administered to a patient, the neural stem cells and pharmaceutically acceptable carrier can be sterile. Water is a useful carrier when cells are administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as glucose, lactose, sucrose, glycerol monostearate, sodium chloride, glycerol, propylene, glycol, water, ethanol and the like. The composition may also contain minor amounts of wetting or emulsifying agents or pH buffering agents as required. The composition may advantageously take the form of a solution, emulsion, sustained release formulation or any other form suitable for use. The selection of a suitable carrier is within the skill of one of ordinary skill in the art.

  Various neuronal subtypes can be obtained from manipulation of neural stem cells grown in culture. Thus, based on the disclosed methods, specific neuronal subtypes can be isolated and purified from other unrelated or unwanted cells to improve results as needed for the treatment of cognitive impairment Can be used for

  The NSCs in the disclosed methods are derived from one site and can be transplanted to another site within the same subject as the autograft. Furthermore, the NSCs in the disclosed methods are derived from genetically identical donors and can be transplanted as syngeneic grafts. Still further, the NSCs in the disclosed method are derived from genetically non-identical members of the same species and can be transplanted as allografts. Alternatively, NSCs can be derived from non-human sources and transplanted as xenografts. With the development of potent immunosuppressive agents, allogeneic and xenografts of non-human neural precursors such as porcine-derived neural precursors can be grafted onto human subjects.

  The sample tissue can be dissociated by any standard method. In one embodiment, the tissue is dissociated by gentle mechanical trituration using a pipette and a divalent cation-free buffer (eg, saline) to form a suspension of dissociated cells. Sufficient dissociation to obtain mostly single cells is desirable to avoid excessive local cell density.

  For successful commercial application of NSCs, it is desirable to maintain a robust and constant culture with stable growth and differentiation potential through multiple serial passages. As mentioned above, the culture method is optimized to achieve long-term stable growth of individual cell lines of NSCs derived from different areas and ages of CNS development while maintaining its distinct progenitor cell properties be able to. In one embodiment, the stem cells are US Pat. Nos. 8,460,651, 8,236,299, 7,691,629, 5, which are incorporated herein by reference in their entirety. , 753,506, 6,040,180 or 7,544,511.

  In one embodiment, the NSCs of the disclosed methods can include cells that have been previously differentiated for transplantation. For maximum cell yield and simplification of the procedure, confluent cultures that mainly contain a population of undifferentiated cells are harvested for transplantation. However, it should be understood that due to the increased cell density, there may be a small population of cells that has just started to differentiate.

  In one embodiment, the NSCs are concentrated in a solution such as the clinically usable hibernation or freezing solution described above. In one embodiment, the NSCs are concentrated to a suitable cell density that may be the same or different from the cell density for administration of the cells. In one embodiment, the cell density for administration ranges from about 1,000 cells per microliter to about per microliter, depending on factors such as injection site, minimum dose required for beneficial effects, and toxic side effects considerations. It can vary with 1,000,000 cells.

  Due to the low cell survival of donor cells using known methods, delivery of large amounts of cells to a relatively small area was necessary to attempt effective therapy. However, the injection volume is the hydrostatic pressure exerted on the host tissue, and the long injection time associated with the high injection volume exacerbates the risk of surgery. Furthermore, excessive injection of donor cells leads to compression and subsequent damage of the host parenchyma. Known methods have required the preparation of high cell density suspensions for injection in an attempt to compensate for volume constraints. However, high cell density promotes robust cluster formation of transplanted cells, inhibits cell migration or spread, prevents effective treatment beyond a limited area, and impairs smooth integration into host tissues.

  In contrast, fewer cells are required per injection as a result of improved in vivo survival of cells prepared by the disclosed methods. In fact, it was found that there were up to 3-4 times the number of cells injected 6 months after the time of injection, and substantial quantitative survival was demonstrated using the disclosed method. Also, for quantitative survival, reproducible administration of the desired cell dose can be achieved. Thus, in one embodiment, NSCs are concentrated to a density of about 1,000 to about 1,000,000 cells per microliter. In one embodiment, the NSCs are concentrated to a density of about 2,000 to about 80,000 NSCs per microliter. In another embodiment, about 5,000 to about 50,000 NSCs per microliter were used for effective engraftment. In another embodiment, about 10,000 to 30,000 NSCs are used per microliter. In a preferred embodiment, NSCs are concentrated to a density of about 70,000 NSCs per microliter.

  In another embodiment, the NSCs are from about 1,000 to about 10,000 cells per microliter, from about 10,000 to about 20,000 cells per microliter, from about 20,000 to about 20,000 per microliter. 30,000 cells, about 30,000 to about 40,000 cells per microliter, about 40,000 to about 50,000 cells per microliter, about 50,000 to about 60,000 per microliter 000 cells, about 60,000 to about 70,000 cells per microliter, about 70,000 to about 80,000 cells per microliter, about 80,000 to about 90,000 cells per microliter Cells, or concentrated to a density of about 90,000 to about 100,000 cells per microliter That.

  In another embodiment, the NSCs are about 100,000 to about 200,000 cells per microliter, about 200,000 to about 300,000 cells per microliter, about 300,000 cells per microliter. To about 400,000 cells, about 400,000 to about 500,000 cells per microliter, about 500,000 to about 600,000 cells per microliter, about 600,000 per microliter From about 700,000 cells, from about 700,000 to about 800,000 cells per microliter, from about 800,000 to about 900,000 cells per microliter, about 900, per microliter It is concentrated to a density of 000 to about 1,000,000 cells.

  In another embodiment, NSCs can be delivered to the treatment area suspended in an injection volume of less than about 100 microliters per injection site. For example, in the treatment of cognitive impairment in human subjects where multiple injections can be made, injection volumes of 0.1 and about 100 microliters per injection site can be used. In a preferred embodiment, the NSC can be delivered to the treatment area suspended in an injection volume of about 1 microliter per injection site.

  In one embodiment, the disclosed method comprises from about 1,000 to about 10,000 cells per microliter, from about 10,000 to about 20,000 cells per microliter, about 20, 000 to about 30,000 cells, about 30,000 to about 40,000 cells per microliter, about 40,000 to about 50,000 cells per microliter, about 50 per microliter , 000 to about 60,000 cells, about 60,000 to about 70,000 cells per microliter, about 70,000 to about 80,000 cells per microliter, about per microliter 80,000 to about 90,000 cells or about 90,000 to about 100,000 per microliter In the cell density of the cells, including the injection of NSC in one or more regions of the brain of the subject.

  In some embodiments, the disclosed methods can include from about 100,000 to about 200,000 cells per microliter, from about 200,000 to about 300,000 cells per microliter, per microliter. About 300,000 to about 400,000 cells, about 400,000 to about 500,000 cells per microliter, about 500,000 to about 600,000 cells per microliter, microliter About 600,000 to about 700,000 cells per microliter, about 700,000 to about 800,000 cells per microliter, about 800,000 to about 900,000 cells or microliter per microliter Cell density of about 900,000 to about 1,000,000 cells per liter , It involves injection of the NSC to one or more regions of the brain of the subject.

  In one embodiment, the disclosed method comprises injecting NSC at a cell density of about 5,000 to about 50,000 cells per microliter. In a preferred embodiment, the disclosed method comprises injecting NSC at a cell density of about 70,000 cells per microliter.

  In one embodiment, the disclosed methods are about 4,000 to about 40,000 cells, about 40,000 to about 80,000 introduced into one or more regions of the subject's brain. Cells, about 80,000 to about 120,000 cells, about 120,000 to about 160,000 cells, about 160,000 to about 200,000 cells, about 200,000 About 240,000 cells, about 240,000 to about 280,000 cells, about 280,000 to about 320,000 cells, about 320,000 to about 360,000 cells Including multiple injections of NSC with cells or a total cell number of about 360,000 to about 400,000 cells.

  In some embodiments, the disclosed methods are about 400,000 to about 800,000 cells, about 800,000 to about 1 introduced into one or more regions of the subject's brain. , 200,000 cells, about 1,200,000 to about 1,600,000 cells, about 1,600,000 to about 2,000,000 cells, about 2,000,000 About 2,400,000 cells, about 2,400,000 to about 2,800,000 cells, about 2,800,000 to about 3,200,000 cells, about 3 , 200,000 to about 3,600,000 cells, or multiple injections of NSC using a total cell number of about 3,600,000 to about 4,000,000 cells.

  The volume of medium in which the NSC grown for delivery to the treatment area is suspended may be referred to herein as the injection volume. The injection volume varies depending on the site of injection and the degenerative state of the tissue. More specifically, the lower limit of the injection volume can be determined by the actual liquid handling of the high cell density viscous suspension as well as the tendency of the cells to cluster. The upper limit of the injection volume can be determined by the limit of compressive force exerted by the injection volume necessary to avoid damaging the host tissue as well as the actual operation time.

  In the disclosed methods, any suitable device for injecting cells into the desired area can be used. In one embodiment, a syringe is used that is capable of delivering sub-microliter volumes over a period of time at a substantially constant flow rate. Cells can be loaded into the device by needles or flexible tubes or any other suitable transfer device.

  In another embodiment, the cells are injected between about 2 to about 5 sites in the brain. In one embodiment, the cells are injected between about 5 to about 10 sites in the brain. In one embodiment, the cells are injected between about 10 to about 30 sites in the brain. In one embodiment, the cells are injected between about 10 to about 50 sites in the brain. The at least two sites can be separated by a distance of approximately 100 microns to about 5,000 microns. In one embodiment, the distance between injection sites is about 400 to about 600 microns. In one embodiment, the distance between injection sites is about 100 to about 200 microns, about 200 to about 300 microns, about 300 to about 400 microns, about 400 to about 500 microns, about 500 to about 600 microns, about 600 to About 700 microns, about 700 to about 800 microns, about 800 to about 900 microns, or about 900 to about 1,000 microns. In one embodiment, the distance between injection sites is about 1,000 to about 2,000 microns, about 2,000 to about 3,000 microns, about 3,000 to about 4,000 microns, or about 4,000 to About 5,000 microns. The distance between injection sites is based on the generation of substantially uninterrupted, continuous donor cells present in the spinal cord tissue and achieves survival of about 2-3 months in animal models such as rats or pigs. It can be determined based on the average volume of demonstrated injections. The actual number of injections in humans and the distance between injections can be estimated from results in animal models.

  The NSC of the disclosed method can generate a large number of neurons in vivo. If NSCs are not clearly pre-differentiated prior to transplantation, NSCs can grow up to 2-4 cell divisions in vivo prior to differentiation, thereby reducing the number of effective donor cells. Further increase. Neurons secrete specific neurotransmitters when differentiated. In addition, neurons secrete growth factors, enzymes and other proteins or substances that are beneficial for various conditions into the environment surrounding the graft in vivo. Thus, because of the ability of the implanted cells to generate a large number of neurons in vivo, and cognitive dysfunction can be caused by the loss of elements, including neuron-derived elements, or the consequence of that loss As such, various conditions can be treated by the disclosed methods. Thus, subjects suffering from cognitive impairment due to the absence of such neuron-derived elements such as growth factors, enzymes and other proteins can be effectively treated by the disclosed methods.

  In one embodiment, the composition comprising an amount of NSC is administered intramuscularly, intraperitoneally, intracerebral, intravenously, subcutaneously, intraarticularly, by intravenous administration, for example, as a bolus or by continuous infusion over a period of time. It can be administered to a subject according to known methods, such as by the sac or by the intrathecal route. Intramedullary, intrathecal, intravenous, intraperitoneal or subcutaneous administration of cells is preferred, and intracerebral, intrathecal or intravenous routes are particularly preferred. However, other cell administration paradigms well known in the art can also be used.

  In one embodiment, the NSC compositions of the present invention are formulated as injectable formulations and include, for example, aqueous solutions or suspensions of active ingredients suitable for intracerebral delivery. When preparing a composition for injection, particularly intracerebral delivery, less than about 7, or less than about 6, such as about 2 to about 7, about 3 to about 6, or about 3 to about 5. There may be a continuous phase comprising an aqueous solution of a tension modifier buffered to pH. Tension modifiers can include, for example, sodium chloride, glucose, mannitol, trehalose, glycerol, or other agents that provide osmotic pressure of a formulation isotonic with blood. Alternatively, if a large amount of tension modifier is used in the formulation, it can be diluted prior to injection with a pharmaceutically acceptable diluent to provide an isotonic mixture with blood.

  In some embodiments of any of the above methods, the composition comprising NSC is administered once. In some embodiments of any of the above methods, administration of the first dose, a composition comprising NSC, is followed by administration of one or more subsequent doses. As an example of a dosing regimen that can be used in the methods of the present disclosure (eg, an interval between a first dose and one or more subsequent doses), an interval of about once a week to about once every 12 months About once every two weeks, about once every six months, about once every month, about once every six months, about once every one month, about once every three months, or 3 An interval of about once a month to about once every 6 months can be mentioned. In some embodiments, administration is monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, or upon disease recurrence.

  In one embodiment, the NSC is injected between about 5 to about 50 sites. In one embodiment, the NSC is injected between about 10 to about 30 sites. At least two of the sites can be separated by a distance of approximately 100 microns to about 5000 microns. In one embodiment, the distance between injection sites is about 400 to about 600 microns. The actual number of injections in humans can be estimated from results in animal models.

  The methods of the present disclosure can include administration of one or more immunosuppressive drugs before, with, or after the injection of NSCs.

  In some embodiments, the NSC and the immunosuppressant drug can be co-administered. The NSCs and immunosuppressive drugs that make up the therapy may be a combined dosage form or a separate dosage form intended for substantially simultaneous administration. NSCs and immunosuppressive drugs can also be administered sequentially, with either NSCs or immunosuppressive drugs being administered by a regimen requiring multiple step administration. Thus, the regimen may require sequential administration of NSCs and immunosuppressive drugs using spaced administration of separate active agents. The duration between multiple administration steps depends on the characteristics of the NSCs and immunosuppressive drugs, such as the efficacy, solubility, bioavailability, plasma half-life and kinetic profile of the therapeutic compound, and the effect and age and condition of the subject's oral food intake Depending on, for example, it can vary from minutes to hours to days. Circadian variation of the target molecule concentration can also influence the optimal dose interval. Whether administered at the same time, substantially simultaneously or sequentially, NSCs and immunosuppressive drugs are, for example, NSCs by the intravenous route, and by the oral, transdermal, intravenous, intramuscular route, or A regimen may be required that requires administration of an immunosuppressive drug by direct absorption by mucosal tissue. Neural stem cells and immunosuppressive drugs, separately or together, orally, by inhalation spray, rectally, topically, buccal (eg sublingual), or parenterally (eg subcutaneously, intramuscularly Intravenous and intradermal injection or infusion techniques), each such therapeutic compound is administered as a pharmaceutically acceptable excipient, diluent or other formulation component. Contained in a suitable pharmaceutical formulation.

  Without further explanation, one of ordinary skill in the art would use the foregoing description and the following illustrative examples to make and utilize the agents of the present disclosure and perform the claimed methods. The following working examples are provided to facilitate the implementation of the present disclosure and should in no way be construed as limiting the remainder of the present disclosure.

  The invention is further illustrated by the following examples, which should in no way be construed as limiting. The materials and methods as used in the following experimental examples are described below.

Example 1
(Materials and methods)
(Preparation of HK532)
Human HK532 NSC strains (NSI-HK532 and NSI-HK532.UbC-IGF-I) are available from Neurostem, Inc. (Rockville, MD). Briefly, HK532 was prepared from cortical tissue obtained from an 8 week old human fetus after artificial abortion. Materials are from Neuralstem, Inc. And informed consent was made in accordance with National Institutes of Health (NIH) and FDA guidelines. The guidelines were reviewed and approved by an external independent review board as described (Johe et al. (1996) Genes Dev., 10 (24): 3129-40). Cortical NSCs were conditionally immortalized using retroviral vectors containing an immortalizing gene and a neomycin resistance gene. The immortalizing gene contained human c-myc cDNA fused at the 3 'end with a cDNA fragment encoding the C-terminal ligand binding domain of the human estrogen receptor. Cells were selected for neomycin resistance and grown as a single cell line (HK532). The cell line was then transduced with a replication defective recombinant lentiviral vector to induce expression of human IGF-I driven by the human ubiquitin C (UbC) promoter. The resulting cells were grown as a single cell line and were not further selected (HK532.UbC-IGF-I). Transduction of HK532 using a control construct expressing green fluorescent protein (GFP) under the same UbC promoter resulted in approximately 90-95% GFP positive proliferating cells.

(HK532 culture and differentiation)
Culture of both HK532 and HK532-IGF-I cells was performed as described previously [42]. Briefly, cells are treated with 100 μg / mL poly-D-lysine (Millipore, Billerica, Mass.) In 10 mM Hepes buffer for 24 hours, followed by 25 μg / mL fibronectin in PBS. Grow on flasks coated for 1 hour. Alternatively, cells were seeded on poly-L-lysine coated inserts prior to co-culture with cortical neurons (CN). Cells were cultured in N2B + medium (supplied by Neurostem, Inc., Rockville, MD) supplemented with 10 ng / mL fibroblast growth factor (FGF) for progenitor cell state growth and maintenance. For differentiation, cells were cultured in NSDM differentiation medium without FGF (4 mM L-glutamine, 20 μM L-alanine, 6 μM L-asparagine, 67 μM L-proline, 250 nM vitamin B12, 25 mg / L insulin, DMEM supplemented with 100 mg / L transferrin, 20 nM progesterone, 100 μM putrescine and 30 nM sodium selenite. Differentiated cell data is shown as the number of days after differentiation (ie, undifferentiated (D0), day 1 (D1), day 3 (D3), etc.). The medium was changed every 2 days, and 50% of the medium was changed.

(IGF-I production and signal transduction)
In HK532 and HK532-IGF-I cells, IGF-I expression and signaling was examined by ELISA and Western blotting as previously described (Vincent et al., Endocrine Society Abstracts (2003), P3-316 548; And Chia et al., Am J Epidemiol (2008), 167 (12): 1438-45). Briefly, to confirm IGF-I production, conditioned media from undifferentiated (D0) and differentiated (D3 and D7) HK532 and HK532-IGF-I cells was collected and Centricon filters (3KDa cut-off, Millipore, Millipore, Billerica, MA) was concentrated 10-fold to 1 mL and a human specific IGF-I ELISA (Assay Designs, Enzo Life Sciences Inc., Farmingdale, NY) was performed according to the manufacturer's instructions. For IGF-I signaling analysis, HK532 and HK532-IGF-I cells were cultured for 4 hours in treated medium (NSDM differentiation medium without added insulin), followed by addition of a selective inhibitor for 1 hour. Then, exogenous IGF-I (20 nM) was added for 30 minutes. Inhibitors include Akt pathway inhibitor LY294002 (LY; 20 μM; Sigma-Aldrich, St. Louis, MO), MAPK inhibitor U0126 (U; 20 μM; Calbiochem, La Jolla, Calif.) Or IGF-IR inhibitor NVPAEW541 (NVP) 1 μM; Sigma-Aldrich). For Western blot, ice-cold RIPA buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1 mM Na deoxycholate, 1% Triton X-100, 0.1 trypsin unit / L Total cell protein was extracted in aprotinin, 10 mg / mL leupeptin and 50 mg / mL PMSF), protein concentration was determined, samples were electrophoresed on SDS-PAGE gels and transferred to nitrocellulose. Primary antibodies (obtained from Cell Signaling Technology, Inc. (Danvers, Mass.) Unless otherwise noted) are phospho-IGF-IR (pIGF-IR), IGF-IRβ (Tyr1135 / 1136), phospho-Akt. (Ser473) (pAkt), Akt, phospho-ERK (pERK), ERK and β-actin (Chemicon, Temecula, CA). After incubating the primary antibody at 4 ° C. overnight, the membrane is incubated for 1 hour at 22 ° C. with a suitable secondary antibody conjugated with horseradish peroxidase (Cell Signaling Technology, Danvers, Mass.) To produce a chemiluminescent substrate. Color was developed using (SuperSignal West Pico; Pierce, Fisher Scientific, Hampton, NH) and exposed to Kodak BioMax XAR film (Sigma-Aldrich).

(Cell migration)
Undifferentiated HK532 and HK532-IGF-I cells are stored at 4 ° C. overnight (1 × 10 6 cells / mL or 3 × 10 6 cells / vial) and then added to the migration insert or alternatively 6 Cultured on well plates and transferred to inserts at D7 of differentiation. NSDM and 10% FBS with or without IGF-I (final concentration of 10 nM) were added below the insert. After 24 hours, cells that migrated through the insert were stained using the QCM 24-well colorimetric cell migration assay (Millipore). Migration was quantified using a standard LabSystems Fluoroskan Ascent FL microplate reader at 530 and 590 nm.

(Cell proliferation and differentiation)
Cell proliferation and differentiation was assessed using a standard laboratory immunocytochemistry (ICC) protocol (Kim et al., Journal of Biological Chemistry (1997), 272: 21268-21273; Lunn et al., Neurobiol Dis (2012). Year), 46 (1): 59-68). Briefly, HK532 and HK532-IGF-I cells were cultured on poly-L-lysine and fibronectin coated glass coverslips in 24-well plates. Cell proliferation was determined by incubating the cells with 10 μM 5′-ethynyl-2′-deoxyuridine (EdU) for 2 hours, followed by fixation and treatment according to the manufacturer's protocol for the Click-It EdU kit (Invitrogen). Measured as previously described at D0, D3 and D7 [45]. EdU incorporation was measured by quantification of fluorescence images taken using an Olympus BX-51 microscope equipped with a digital camera. Approximately 2.5-2.7 × 10 3 cells per proliferation experiment were counted for all samples (n = 3).

  To assess differentiation, cells were fixed with 4% PFA, permeabilized with 0.1% Triton / PBS, and blocked in 5% normal donkey serum / 0.1% Triton / PBS. Next, Ki67 (Novus, Littleton, CO), TUJ1 (Neuromics, Edina, MN), Nestin (Chemicon, Millipore), GAD65 / 67 (Millipore), VGLUT2 (Millipore) or IGF-IRgma (1 :) Antibodies were incubated at 1: 1000 overnight at 4 ° C. unless otherwise noted. Cells are then incubated in a secondary antibody (Jackson ImmunoResearch, Westgrove, PA) conjugated with Cy3, Cy5 or FITC, followed by ProLong Gold antifade (Molecular Probes, Invitrogen, Carlsbad) with DAPI. Was mounted on a glass slide. Images were taken using an Olympus BX-51 microscope and approximately 2.5-2.7 × 10 3 cells were counted per differentiation experiment for all samples (n = 3).

  We then use our established neural index measurements as described previously to demonstrate the effect of induced IGF-I expression on progenitor cell state maintenance and axonal outgrowth. (Lunn et al., Stem Cells Dev (2010), 19 (12): 1983-93). Briefly, cells are cultured in monolayers on glass coverslips for the first 7 days of differentiation and immunolabeled with Nestin at D0, D3 and D7 to identify neural progenitor cells or TUJ1 And immunolabeled to observe primary neuronal processes. All Nestin labeled samples were counted over 2.5 × 10 3 cells per experiment (n = 3). Alternatively, MetaMorph (Molecular Devices, Sunnyvale, CA) was used to analyze TUJ1-labeled images and their corresponding DAPI images. A threshold was set and the area covered by neurites was measured using area statistics. Cell counts were counted using the “Count Nucleus” plug-in and manual adjustments were made to correct any miscounted cells. Compound nerve index measurements were used to analyze the number of neurons and the length of neurites, which are expressed as the complete neuronal area divided by the number of nuclei. Data are presented as neurite area per cell (μm2 / cell) (Lunn et al., Stem Cells Dev (2010), 19 (12): 1983-93). A total of 6 images per state are counted, which corresponds to approximately 7.5 × 10 3 DAPI-labeled cells (n = 3).

(Evaluation of primary CN preparation and neuroprotection)
Primary CN was isolated according to our previously published protocols (Lunn et al., Stem Cells Dev (2010), 19 (12): 1983-93). Briefly, CN obtained from E15 Sprague-Dawley rat embryos was collected, the membrane was removed, and the tissue was minced into 2-3 mm pieces. Cells were dissociated by incubating the tissue in 0.5% trypsin / EDTA at 37 ° C. for 10 minutes, followed by trituration for 1 minute using a serum-coated glass pipette. The resulting cell suspension was spread on a poly-L-lysine coated glass coverslip in a 24-well plate and 2.5 mg / ml albumin, 2.5 μg / ml catalase, 2.5 μg / ml SOD, 0.01 mg / ml transferrin, 15 μg / ml galactose, 6.3 ng / ml progesterone, 16 μg / ml putrescine, 4 ng / ml selenium, 3 ng / ml β-estradiol, 4 ng / ml hydrocortisone, 1 × penicillin / streptomycin / neomycin Incubated in growth medium containing Neurobasal medium (Gibco BRL, Invitrogen) supplemented with (Gibco BRL) and 1 × B-27 additive (Gibco BRL).

  To examine CN, HK532, and HK532-IGF-I sensitivity to the toxic AD microenvironment, cells were treated with 10 μM Aβ (1-42) (rPeptide) for approximately 72 hours. Cell damage was assessed by cleaved caspase 3 activation (CC3), determined by counting the percentage of CC3 positive cells after ICC. Approximately 2.5 × 10 3 cells per experiment were counted for all samples (n = 3). To assess the neuroprotective effect, differentiated D7 HK532-IGF-I was seeded on the insert and co-cultured with the primary CN culture in normal growth medium. After 24 hours, co-cultures were treated with 10 μM Aβ (1-42) (rPeptide) for 72 hours. Primary CN was immobilized for ICC analysis as described above using CC3 antibody (1: 1000, Cell Signaling).

(In vivo transplantation)
To demonstrate that HK532-IGF-I cells survive and integrate into the hippocampal region after in vivo transplantation, from Jackson Laboratory (Bar Harbor, ME), 6 weeks old B6C3-Tg (APPswe / PSEN1ΔE9) 85Dbo / J (APP / PS1; n = 5) and wild type B6C3F1 / J (WT; n = 8) mice were obtained. At 11 weeks of age, mice were given subcutaneous tacrolimus pellets (FK-506; supplied by Neurostem, Inc.) and cell transplant surgery was performed at 12 weeks of age. Briefly, mice were anesthetized with isofluorane and placed in a standard stereotaxic frame (Stoelting Company, Wood Dale, IL). An incision was made in the skin and a large craniotomy was performed in the expected area of injection. The following coordinates measured from the cross stitch (rear side / side / ventral side, respectively): −0.82 / ± 0.75 / 2.5, −1.46 / ± 2.3 / 2.9, − The HK532-IGF-I cell suspension was administered by bilateral injection (total 6 injections) into the hippocampal arch at 3 sites expressed according to 1.94 / ± 2.8 / 2.9. Each injection consisted of a volume of 1 μL at a cell concentration of 30,000 cells / μL (administered over 60 seconds, delayed 60 seconds before withdrawing the needle). The skin was then closed using absorbable sutures. Following surgery, mice were given intraperitoneal narcotic analgesics for 2 days and continued with tacrolimus pellets throughout the study. Mice were sacrificed for analysis at 2 and 10 weeks after cell transplantation. Briefly, animals were anesthetized, perfused with ice-cold saline, the brain was dissected and an incision was made along the interhemispheric boundary. Brains were post-fixed overnight in 4% PFA and cryoprotected in 30% sucrose for immunohistochemical analysis (IHC).

  For IHC, fixed brain tissue was embedded using Optimal Cutting Temperature Compound (OCT) and 14 μm slices were sectioned using cryostat. Ten sections of the hippocampus were selected per animal for IHC, grafted cells were detected, and correct targeting to the hippocampal arch was confirmed. Sections were rehydrated with PBS, permeabilized in 0.5% Triton X-100 in PBS for 20 minutes, and blocked in 5% donkey serum in 0.1% Triton X-100 in 1X PBS for 30 minutes. did. Primary antibodies against doublecortin (DCX; Millipore) and human nuclei (HuNu; Millipore) were diluted 1: 200 with blocking solution and incubated with sections overnight at 4 ° C. After incubating the primary antibody, the sections were washed three times in PBS and incubated with fluorescent conjugated secondary antibodies (Alexa 488 and Alexa 594; 1: 500; Invitrogen) made in donkey for 1 hour. Once staining was complete, the slides were mounted with glass coverslips using ProLong Gold anti-fade mounting media containing DAPI nuclear stain (Molecular Probes, Invitrogen). Fluorescence images were taken using a Leica SP2 confocal microscope.

(Statistical analysis)
All data are shown as mean ± standard deviation (SD), n = 3, or as representative images of 3 independent experiments. Statistical analysis was performed using GraphPad Prism (GraphPad Software, Inc., La Jolla, Calif.). A paired t test was used for pairwise comparisons. A value of p <0.05 was considered statistically significant (* p <0.05).

(Example 2)
(IGF-I production and signal transduction)
HK532 cells are a novel cortical NSC not previously described. To enhance their potential efficacy as cell therapeutics and to examine the effects of IGF-I production on their neuroprotective capacity, using lentiviral vectors encoding full-length human IGF-I The HK532-IGF-I cell line was generated. ELISA analysis of the conditioned medium shows that parental HK532 cells produce very little to undetectable basal levels of IGF-I, whereas HK532-IGF-I cells are 3-5 ng / mL between D0 and D7. An approximately 50-fold increase in production of IGF-I (FIG. 1A) was demonstrated. Thus, HK532-IGF-I cells produce appropriate levels of IGF-I, which are maintained throughout early differentiation, confirming robust and stable IGF-I expression.

  An investigation was conducted to see how autocrine IGF-I expression regulates growth factor receptor levels and activation. IGF-IR expression was observed along the cell surface of both D7 parental HK532 and HK532-IGF-I by IHC (FIG. 1B). In both cell lines, this expression was confirmed by Western blot analysis and also showed a significant increase in receptor expression after differentiation (FIG. 1C). Although slightly reduced IGF-IR expression levels were observed in HK532-IGF-I versus HK532 at D0 and D7, IGF-IR phosphorylation and signaling activation were not significantly different between cell lines and Addition of sex IGF-I resulted in increased receptor phosphorylation (FIG. 1C); this activation was significantly more evident after differentiation.

  Given that IGF-I signaling activates mitogen-activated extracellular signal-regulated kinase / extracellular signal-regulated kinase (MEK / ERK), mitogen-activated protein kinases (in HK532 and HK532-IGF-I cells) MAPK) and phosphatidylinositol 3-kinase (PI3K) / Akt pathways, phosphorylation and downstream activation of these critical pathways were evaluated (FIG. 1C). There was basal MAPK signaling at D0 and D7, but there was no significant difference between cell lines, and only IGF-I stimulation increased signaling after differentiation. However, basal Akt signaling was greatly increased in D0 HK532-IGF-I cells and in both cell lines after differentiation. The addition of exogenous IGF-I promoted a robust increase of Akt in post-differentiation HK532 cells, but very little Akt activation was observed after IGF-I stimulation in HK532-IGF-I. These data demonstrate a reduction in the reactivity of HK532-IGF-I to exogenous IGF-I against the parental cells. Of note, inhibition of MAPK signaling increases Akt phosphorylation, and inhibition of Akt signaling results in the opposite observation, indicating that these pathways can be signaled compensably. Suggest. However, inhibition of IGF-IR signaling using the receptor antagonist NVP did not affect MAPK signaling, but significantly depleted IGF-IR and Akt activation below basal levels in both cell lines (FIG. 1C). Together, these data demonstrate that HK532-IGF-I cells exhibit normal IGF-IR and MAPK / Akt signaling profiles.

(Example 3)
(IGF-I expression does not alter HK532 proliferation or migration)
EdU integration was used to assess the effect of IGF-I on HK532 and HK532-IGF-I cell proliferation. Approximately 36% and 33% of untreated D0 HK532 and HK532-IGF-I were EdU positive, respectively (FIGS. 2A-E). At D3, 6% and 9% of HK532 and HK532-IGF-I were EdU positive, respectively, and by D7, less than 3% of either cell line was EdU positive. Therefore, no difference in growth profile was observed with D0, D3 or D7, and both strains showed minimal growth at D7. These data demonstrate that IGF-I does not promote or maintain proliferation during the initial stages of differentiation.

  The effect of IGF-I on HK532 and HK532-IGF-I migration was also evaluated at D0 and D7. Equivalent migration levels were observed for HK532 and HK532-IGF-I at both time points (FIGS. 2F-G). Furthermore, no change was observed when additional IGF-I was added below the transwell insert. Thus, induced IGF-I expression did not exert a discernable effect on progenitor cell migration.

Example 4
(HK532 expressing IGF-I retains neuronal differentiation ability)
Next, the effect of IGF-I on maintaining HK532 progenitor state and axonal outgrowth was examined. Approximately 92% and 90% of D0 HK532 and HK532-IGF-I, respectively, are Nestin positive, indicating that IGF-I expression did not affect maintenance of the progenitor cell state. The effect of IGF-I on neurite outgrowth was also evaluated using a neural index approach established as an initial indicator of neuronal differentiation. For both HK532 and HK532-IGF-I cells, as the cells differentiated, the nerve index increased between D0 and D7, and no difference was observed between the cell lines at any time point tested (FIG. 3F). These data demonstrate that IGF-I does not affect early HK532 differentiation.

(Example 5)
(HK532-IGF-I has an increased phenotype that is GABAergic and not glutamatergic)
Percentage of cells exhibiting glutamatergic (VGLUT) and GABAergic (GAD65) phenotypes at D0, D3 and D7 to examine the effect of IGF-I on terminal differentiation. GAD65 positive cells were quantified and increased significantly in HK532-IGF-I compared to parental HK532 cells in 74% and 67% of total cells, respectively (FIGS. 4A, B, E). The percentage of VGLUT positive cells in HK532 (61%) and HK532-IGF-I (67%) cultures was not significantly different (FIGS. 4C, D, F). These data demonstrate that the presence of IGF-I increases the number of GABAergic neurons due to cell differentiation but has no significant effect on the number of glutamatergic neurons.

(Example 6)
(HK532-IGF-I is resistant to Aβ toxicity in vitro and protects primary CN)
Aβ (1-42) is a commonly used in vitro model of AD-related toxicity (Bruce et al. (1996), PNAS 93 (6): 2312-6). Significant apoptosis and CC3 activation was observed in primary CN and both NSC lines when exposed to Aβ (FIG. 5A). The level of apoptosis in HK532 and HK532-IGF-I was significantly lower than that observed in primary CN (p <0.05; FIG. 5A). To investigate the protective ability of the modified progenitor cells, Aβ toxicity was also evaluated in primary CN indirectly co-cultured with HK532 and HK532-IGF-I (FIGS. 5B-E). Apoptosis in primary CN, also indicated by CC3 activation, was greatly reduced to less than 40% when co-cultured with HK532 and less than 30% when co-cultured with HK532-IGF-I (p <0 .05; FIG. 5F). These data indicate that the HK532-IGF-I cell line is neuroprotective and can prevent Aβ-induced primary CN death.

(Example 7)
(HK532-IGF-I survives transplantation and is integrated in vivo in the AD mouse model)
To establish the feasibility of preclinical studies, HK532-IGF-I cells were transplanted into APP / PS1 double transgenic mice, a commonly used model of AD (Cao et al., J Biol Chem (2007 ), Volume 282 (No. 50): 36275-82). This pilot study worked to confirm the correct and valid anatomical placement of cells in the hippocampal hippocampus of the hippocampus and evaluated the survival of the transplanted cells over time. Targeting accuracy was achieved in all injected animals. Transplanted human cells were detected by IHC for HuNu and DCX at 2 weeks (data not shown) and 10 weeks after transplantation (FIGS. 5E, F). Grafted cells were evident in the hippocampal region of both ADs (FIG. 5E) and WT animals (FIG. 6F). Co-staining of HuNu labeled cells with DCX, a microtubule-associated phosphorylated protein that labels neuronal progenitors, indicates neurogenesis, suggesting that the transplanted cortical progenitor cells were in the early neuronal differentiation phase.

(Example 8)
(Administration of HK532-IGF-I reduces Aβ plaque formation in vivo in an AD mouse model)
In order to evaluate the overall effect of in vivo HK532-IGF-I transplantation on Aβ pathology, multiple antibodies per mouse were used with polyclonal antibodies against five Aβ isoforms (Aβ-37, 38, 39, 40 and 42). Immunostaining was performed on hippocampal and cortical sections. Based on the scale and intensity of the total immunoreactive area, the fluorescence images of the sections were quantified. As expected, the results show clear Aβ plaque formation in non-tg animals in APP / PS1 mice injected with vehicle and no Aβ (FIGS. 6A-B, D-E). Furthermore, APP / PS1 mice treated with HK532-IGF-I had a significantly significant reduction in Aβ levels compared to vehicle / injected APP / PS1 mice (P <0.0001; Unpaired t-test) (FIGS. 6B-C, EG). This data indicates that HK532-IGF-I not only functions to protect neural tissue from Aβ-induced damage, but also eliminates Aβ deposits and / or makes Aβ less likely to accumulate. Indicates that the

  To gain more insight into the mechanism of Aβ reduction in NSC-treated mice, differences in Aβ levels in the hippocampus and cortex were considered separately. Aβ was significantly reduced in the cortex of NSC-treated mice (P <0.0005; unpaired t-test) (FIG. 6H). However, the reduction of Aβ in the hippocampus of NSC-treated mice was not significant (P = 0.1061; unpaired t-test) (FIG. 6H).

Example 9
(Administration of HK532-IGF-I increases cholinergic activity in vivo in the AD mouse model)
To evaluate the presence of cholinergic neurons in our AD model and to examine the effects of HK532-IGF-I transplantation on these neurons, striatum sections from each mouse were used with antibodies to ChAT. And immunostained to identify strong levels of ChAT expressing cholinergic neurons (FIGS. 7A-D). We imaged the entire striatum for each section and counted the number of each ChAT positive cell. Cell counts showed a significant loss of striatal cholinergic neurons in APP / PS1 mice compared to WT (P = 0.115; unpaired t-test) (FIG. 7E). Furthermore, there was a significant increase in the number of cholinergic neurons in NSC-treated APP / PS1 mice compared to vehicle-injected AD mice (P = 0.0366; unpaired t-test) (FIG. 7F ). These results indicate a rescue of cholinergic function in the striatum by HK532-IGF-I transplantation in APP / PS1 mice.

(Example 10)
(Administration of HK532-IGF-I increases presynaptic activity in vivo in an AD mouse model)
To examine whether HK532-IGF-I increases the density of synapses in APP / PS1 mice, hippocampal sections obtained from all animals were immunostained using the presynaptic marker, synaptophysin. In the hippocampus of APP / PS1 mice transplanted with HK532-IGF-I, a discernable increase in fluorescence intensity was seen compared to transgenic mice injected with vehicle (FIGS. 8A-F). This increased intensity was comparable to the level seen in both uninjected and sham-treated non-tg mice. These data indicate that HK532-IGF-I transplant rescues memory and cognition by modifying synapses in AD.

  To examine whether HK532-IGF-I cells had formed synapses with endogenous neurons, co-staining for human NuMA and synaptophysin of both mouse and human origin was performed on mice treated with HK532-IGF-I. Of hippocampal slices. In the region of the polymorphic layer where NSCs were localized, considerable synaptophysin positive staining was seen (FIG. 8I). Furthermore, synaptic markers are clearly adjacent to NuMA stained cells (FIGS. 8G-J), suggesting that human NSC-derived cells can synapse with endogenous neurons.

(Example 11)
(Survival and integration of HK532.UbC-IGF1 in the spinal cord)
To demonstrate survival and integration in the spinal cord, HK532. UbC-IGF1 was implanted into the cervical spinal cord of an established animal model of SOD1G93A rats, amyotrophic lateral sclerosis (ALS). Each animal was injected with a total of 1.8 × 10 5 cells targeting the anterior horn of the cervical spinal cord (C4-C6 spinal level). Animals were immunosuppressed by transient mycophenolate mofetil (30 mg / kg IP, 7 days after grafting) and by continuous tacrolimus delivery. The animals were alive for 56 days and then standard perfusion-fixed. Cryoimmunohistochemical analysis shows the presence of human cell grafts in the anterior horn and broad distribution in gray matter and white matter (FIGS. 9a-c).

  Unless otherwise indicated, all numbers representing amounts of components, properties such as molecular weight, reaction conditions, etc. used in the specification and claims are modified in all cases by the term “about”. Should be understood. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure. At least not as an attempt to limit the application of the doctrine of claims, but each numerical parameter should be interpreted at least by applying the usual rounding method, taking into account the reported significant digits. Must not.

  Although the numerical ranges and parameters representing the broad scope of this disclosure are approximate, the numerical values shown in the specific examples are reported as accurately as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

  The terms “a”, “an”, “the” and similar designations used in the context of describing the present disclosure (especially in the context of the following claims) Should be construed to include both the singular and the plural unless specifically stated otherwise or otherwise clearly contradicted by context. The description of unit of value herein is intended to serve merely as a concisely expressed way of referring individually to each distinct value falling within the range. Unless stated otherwise specifically in the specification, each individual value is incorporated herein as individually listed herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples or exemplary languages (e.g., "such as") provided herein is intended merely to further clarify the present disclosure and is not separately claimed. It does not impose limitations on the scope of disclosure. No language in the specification should be construed as indicating any non-claimed element required to implement the disclosure.

  Groupings of alternative elements disclosed herein or embodiments of the present disclosure should not be construed as limiting. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that for convenience and / or patentability reasons, one or more members of a group may be included in or removed from the group. In the event of any such incorporation or deletion, this specification shall contain such modified groups that satisfy the description of all Markush groups used in the appended claims. To do.

  Specific embodiments of this disclosure are described herein, including the best mode known to the inventors for carrying out the disclosure. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect those skilled in the art to use such variations as appropriate, and the inventors intend that the present disclosure may be practiced rather than as specifically described herein. To do. Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the disclosure unless otherwise indicated herein or otherwise clearly contradicted by context.

  Particular embodiments disclosed herein may be further limited in the claims using language or consisting essentially of language. When used in the claims, the transitional phrase “consisting of” includes any element, step or ingredient not specified in the claims, whether filed or added for amendment. Exclude. The transitional phrase “consisting essentially of” limits the scope of the claims to those that do not significantly affect the particular material or step and basic and novel feature (s). Embodiments of the present disclosure so claimed are essentially or explicitly described and enabled herein.

  It should be understood that the embodiments of the present disclosure disclosed herein are illustrative of the principles of the present disclosure. Other modifications that may be used are within the scope of this disclosure. Thus, by way of example, and not limitation, alternative arrangements of the present disclosure can be utilized in accordance with the techniques herein. Accordingly, the present disclosure is not limited to that precisely as shown and described.

  Although the present disclosure has been described and illustrated herein with reference to various specific materials, procedures and examples, the present disclosure is limited to the specific combinations of materials and procedures selected for that purpose. It is understood that it is not done. Many variations of such details may be implied as will be appreciated by those skilled in the art. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the disclosure being indicated by the following claims. All references, patents and patent specifications mentioned in this application are hereby incorporated by reference in their entirety.

The present disclosure is a method for restoring memory and / or cognition in a subject comprising a therapeutically effective amount of an exogenous polynucleotide encoding IGF-1 in one or more regions of the subject's brain 1 Or a method comprising administering a plurality of human neural stem cells.
In certain embodiments, for example, the following are provided:
(Item 1)
A stable human neural stem cell comprising an exogenous polynucleotide encoding insulin-like growth factor 1 (IGF-1).
(Item 2)
2. The human neural stem cell of item 1, wherein the human neural stem cell is capable of differentiating into a significantly increased number of GAD65-positive GABAergic neurons as compared to neural stem cells that do not contain an exogenous polynucleotide encoding IGF-1.
(Item 3)
A human neural stem cell comprising an exogenous polynucleotide encoding a growth factor.
(Item 4)
The growth factors include insulin-like growth factor 1 (IGF-1), glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and vascular endothelial cells Item 4. The human neural stem cell according to Item 3, which is a neurotrophic factor selected from the group consisting of growth factors (VEGF).
(Item 5)
Item 5. The human neural stem cell according to item 4, wherein IGF-1 is an IGF-1 isoform.
(Item 6)
Item 6. The human neural stem cell according to Item 5, wherein the IGF-1 isoform is IGF-1 isoform 4.
(Item 7)
Item 5. The human neural stem cell according to Item 4, wherein the IGF-1 isoform 4 comprises the nucleotide sequence represented by SEQ ID NO: 1.
(Item 8)
Item 4. The human neural stem cell according to Item 3, derived from a tissue selected from the group consisting of cortex, hippocampus, thalamus, midbrain, cerebellum, hindbrain, spinal cord and dorsal root ganglion.
(Item 9)
4. The human neural stem cell according to item 3, obtained from a fetus or an embryo.
(Item 10)
10. The human neural stem cell according to item 9, obtained from a fetus having a fetal age of about 5 to about 20 weeks.
(Item 11)
Item 4. The exogenous polynucleotide encoding the growth factor is operably linked to a ubiquitin C (UbC) promoter, a human phosphoglycerate kinase 1 promoter, a human synapsin promoter, or a synthetic CAG promoter. Human neural stem cell.
(Item 12)
A human neural stem cell comprising an exogenous polynucleotide encoding insulin-like growth factor 1 (IGF-1), wherein IGF-1 comprises the nucleotide sequence shown in SEQ ID NO: 1, and the IGF-1 nucleotide sequence is stable Human neural stem cells that are expressed.
(Item 13)
A method for the treatment of a neurodegenerative disease or disorder comprising administering to a subject one or more human neural stem cells comprising a therapeutically effective amount of an exogenous polynucleotide encoding a growth factor, A method wherein the growth factor is stably expressed.
(Item 14)
The growth factors include insulin-like growth factor 1 (IGF-1), glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and vascular endothelial cells 14. The method of item 13, which is a neurotrophic factor selected from the group consisting of growth factors (VEGF).
(Item 15)
15. A method according to item 14, wherein IGF-1 is an IGF-1 isoform.
(Item 16)
16. The method according to item 15, wherein the IGF-1 isoform is IGF-1 isoform 4.
(Item 17)
The method according to item 16, wherein the IGF-1 isoform 4 comprises the nucleotide sequence shown in SEQ ID NO: 1.
(Item 18)
The neurodegenerative disease or disorder is amyotrophic lateral sclerosis (ALS), spinal cord injury (SCI), traumatic brain injury (TBI), Alzheimer's disease (AD), dementia, mild cognitive impairment, diabetes, diabetes 14. The method of item 13, wherein the method is a related CNS complication, peripheral neuropathy, retinal neuropathy or multiple sclerosis.
(Item 19)
19. A method according to item 18, wherein the spinal cord injury is traumatic spinal cord injury or ischemic spinal cord injury.
(Item 20)
A method of reducing amyloid beta (Aβ) deposition in a subject's brain, eliminating Aβ deposits in a subject's brain, or preventing Aβ accumulation in a subject's brain, wherein the method comprises: Administering a therapeutically effective amount of one or more human neural stem cells comprising an exogenous polynucleotide encoding IGF-1.
(Item 21)
21. The method of item 20, wherein the one or more regions of the brain comprise the hippocampus and / or cortex.
(Item 22)
A method of increasing the number of cholinergic neurons in a subject's brain, comprising one or more exogenous polynucleotides encoding IGF-1 in one or more regions of said subject's brain Administering a human neural stem cell.
(Item 23)
24. The method of item 22, wherein the one or more regions of the brain comprise the hippocampus and / or cortex.
(Item 24)
A method of repairing synapses in a brain of a subject, wherein one or more human neural stem cells comprising a therapeutically effective amount of an exogenous polynucleotide encoding IGF-1 in one or more regions of the subject's brain. Administering.
(Item 25)
A method for restoring memory and / or cognition of a subject comprising one or more humans comprising a therapeutically effective amount of an exogenous polynucleotide encoding IGF-1 in one or more regions of the subject's brain Administering a neural stem cell.

Claims (25)

  A stable human neural stem cell comprising an exogenous polynucleotide encoding insulin-like growth factor 1 (IGF-1).   2. The human neural stem cell of claim 1, wherein the human neural stem cell is capable of differentiating into a significantly increased number of GAD65-positive GABAergic neurons as compared to neural stem cells that do not include an exogenous polynucleotide encoding IGF-1.   A human neural stem cell comprising an exogenous polynucleotide encoding a growth factor.   The growth factors include insulin-like growth factor 1 (IGF-1), glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and vascular endothelial cells The human neural stem cell according to claim 3, which is a neurotrophic factor selected from the group consisting of growth factors (VEGF).   The human neural stem cell according to claim 4, wherein IGF-1 is an IGF-1 isoform.   6. The human neural stem cell according to claim 5, wherein the IGF-1 isoform is IGF-1 isoform 4.   The human neural stem cell according to claim 4, wherein the IGF-1 isoform 4 comprises the nucleotide sequence shown in SEQ ID NO: 1.   The human neural stem cell according to claim 3, derived from a tissue selected from the group consisting of cortex, hippocampus, thalamus, midbrain, cerebellum, hindbrain, spinal cord and dorsal root ganglion.   The human neural stem cell according to claim 3, which is obtained from a fetus or an embryo.   10. The human neural stem cell of claim 9, obtained from a fetus having a fetal age of about 5 to about 20 weeks.   4. The exogenous polynucleotide encoding the growth factor is operably linked to a ubiquitin C (UbC) promoter, a human phosphoglycerate kinase 1 promoter, a human synapsin promoter, or a synthetic CAG promoter. Human neural stem cells.   A human neural stem cell comprising an exogenous polynucleotide encoding insulin-like growth factor 1 (IGF-1), wherein IGF-1 comprises the nucleotide sequence shown in SEQ ID NO: 1, and the IGF-1 nucleotide sequence is stable Human neural stem cells that are expressed.   A method for the treatment of a neurodegenerative disease or disorder comprising administering to a subject one or more human neural stem cells comprising a therapeutically effective amount of an exogenous polynucleotide encoding a growth factor, A method wherein the growth factor is stably expressed.   The growth factors include insulin-like growth factor 1 (IGF-1), glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and vascular endothelial cells 14. The method of claim 13, wherein the method is a neurotrophic factor selected from the group consisting of growth factors (VEGF).   15. The method of claim 14, wherein IGF-1 is an IGF-1 isoform.   16. The method of claim 15, wherein the IGF-1 isoform is IGF-1 isoform 4.   17. The method of claim 16, wherein the IGF-1 isoform 4 comprises the nucleotide sequence shown in SEQ ID NO: 1.   The neurodegenerative disease or disorder is amyotrophic lateral sclerosis (ALS), spinal cord injury (SCI), traumatic brain injury (TBI), Alzheimer's disease (AD), dementia, mild cognitive impairment, diabetes, diabetes 14. The method of claim 13, wherein the method is associated CNS complications, peripheral neuropathy, retinal neuropathy or multiple sclerosis.   19. The method of claim 18, wherein the spinal cord injury is traumatic spinal cord injury or ischemic spinal cord injury.   A method of reducing amyloid beta (Aβ) deposition in a subject's brain, eliminating Aβ deposits in a subject's brain, or preventing Aβ accumulation in a subject's brain, wherein the method is applied to one or more regions of the subject's brain Administering a therapeutically effective amount of one or more human neural stem cells comprising an exogenous polynucleotide encoding IGF-1.   21. The method of claim 20, wherein the one or more regions of the brain include the hippocampus and / or cortex.   A method of increasing the number of cholinergic neurons in a subject's brain, comprising one or more exogenous polynucleotides encoding IGF-1 in one or more regions of said subject's brain Administering a human neural stem cell.   23. The method of claim 22, wherein the one or more regions of the brain include the hippocampus and / or cortex.   A method of repairing synapses in a brain of a subject, wherein one or more human neural stem cells comprising a therapeutically effective amount of an exogenous polynucleotide encoding IGF-1 in one or more regions of the subject's brain. Administering.   A method for restoring memory and / or cognition of a subject comprising one or more humans comprising a therapeutically effective amount of an exogenous polynucleotide encoding IGF-1 in one or more regions of the subject's brain Administering a neural stem cell.